Structural analysis of alcohol-dependent activation of GIRKs

GIRK 酒精依赖性激活的结构分析

• 批准号: 8693873
• 负责人: SENYON CHOE
• 金额: $ 37.53万
• 依托单位: ICAHN SCHOOL OF MEDICINE AT MOUNT SINAI
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-07-25 至 2016-04-14
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8693873
• 关键词: Alcohol abuse; Alcohol consumption; Alcohol dependence; Alcohol withdrawal syndrome; Alcoholism; Alcohols; Animal Model; Behavior; Binding; Biology; Brain; Cessation of life; Chemicals; Chromosomes, Human, Pair 1; Complex; Cysteine; Development; Drug Addiction; Electrophysiology (science); Ethanol; Ethanol dependence; Exhibits; Foundations; Future; GIRK2 subunit, G protein-coupled inwardly-rectifying potassium channel; GIRK3 subunit, G protein-coupled inwardly-rectifying potassium channel; GTP-Binding Proteins; Genes; Goals; Grant; Health; Heavy Drinking; Human; Institutes; Ion Channel; Kinetics; Knockout Mice; Knowledge; Lead; Ligands; Mediating; Modeling; Modification; Molecular; Mus; Mutagenesis; Mutation; Neurons; Pain; Paper; Pentobarbital; Pharmacologic Substance; Physiological; Potassium; Potassium Channel; Predisposition; Proteins; Publications; Publishing; Quantitative Trait Loci; Receptor Signaling; Research; Research Institute; Research Proposals; Resolution; Role; Self-Administered; Seminal; Signaling Molecule; Structure; Testing; Texas; United States; Universities; Wild Type Mouse; Withdrawal; Work; alcohol behavior; alcohol effect; alcohol research; alcohol response; austin; base; economic cost; experience; genetic regulatory protein; hypnotic; innovation; insight; interdisciplinary approach; interest; inward rectifier potassium channel; mutant; novel; relating to nervous system; research study; response; sedative; structural biology; tool

DESCRIPTION (provided by applicant): Alcohol (ethanol) consumption alters neural activity in the brain by modulating different types of ion channels. An emerging concept in the field is that some of the physiological effects of ethanol are mediated by direct modulation of ion channels in the brain. One of the targets in the brain for ethanol is the G protein-gated inwardly rectifying potassium (GIRK) channel, which is activated by ethanol. Mice lacking GIRK2 channels exhibit diminished ethanol-induced tolerance to pain and self-administer more ethanol than wild-type mice. Moreover, a quantitative trait loci with a large effect on predisposition to sedative withdrawal, such as from ethanol, was narrowed to a region on chromosome 1 in mice that contains Girk3 gene. GIRK3 knockout mice exhibit less severe sedative-hypnotic withdrawal. Though GIRK2 and GIRK2/3 channels are implicated in ethanol-related behaviors, the molecular mechanism underlying this response is not well understood. Recently, we showed with high-resolution structural studies that alcohols bind directly to hydrophobic pockets of inwardly rectifying potassium channels. Mutations in the alcohol-binding pocket of GIRK2 channels significantly alter ethanol activation. We hypothesize that ethanol binds to hydrophobic pockets in GIRK2/3 channels and facilitate a conformational change that is relayed to the channel's gate and opens the channel. In this research proposal, we plan to use an innovative approach of high-resolution crystallographic studies, structure-based mutagenesis and advanced electrophysiological recordings to investigate this hypothesis. Specifically, we will conduct a structure-function analysis of the ethanol-binding pocket in GIRK2/3 channels (1), solve high-resolution structures of GIRK channels complexed with ethanol to reveal conformational changes in the channel protein that occur with ethanol-dependent gating (2), and elaborate mechanistic models for ethanol-dependent activation of GIRK channels, utilizing single-channel recordings and chemical modification of cysteine-substituted channels (3). Completion of these proposed experiments will reveal the structural basis of ethanol modulation of GIRK channels, which will provide insights into the mechanism of ethanol-modulation of other types of ion channels. Understanding the molecular mechanism underlying ethanol modulation of ion channels could lead to development of novel pharmaceutical agents for treating alcohol-dependence, directly benefiting human health.

描述（由申请人提供）：酒精（乙醇）的消耗通过调节不同类型的离子通道来改变大脑中的神经活动。该领域的一个新兴概念是，乙醇的一些生理效应是通过直接调节大脑中的离子通道来介导的。大脑中乙醇的目标之一是 G 蛋白门控内向整流钾 (GIRK) 通道，该通道会被乙醇激活。缺乏 GIRK2 通道的小鼠表现出乙醇诱导的疼痛耐受性降低，并且比野生型小鼠自我施用更多的乙醇。此外，对镇静剂戒断（例如乙醇戒断）倾向有很大影响的数量性状位点被缩小到含有 Girk3 基因的小鼠 1 号染色体上的区域。 GIRK3 基因敲除小鼠表现出较不严重的镇静催眠戒断症状。尽管 GIRK2 和 GIRK2/3 通道与乙醇相关行为有关，但这种反应背后的分子机制尚不清楚。最近，我们通过高分辨率结构研究表明，醇直接与内向整流钾通道的疏水性口袋结合。 GIRK2 通道的酒精结合口袋的突变显着改变乙醇的激活。我们假设乙醇与 GIRK2/3 通道中的疏水袋结合并促进构象变化，该变化被传递到通道的门并打开通道。在这项研究提案中，我们计划使用高分辨率晶体学研究、基于结构的诱变和先进的电生理记录的创新方法来研究这一假设。具体来说，我们将对 GIRK2/3 通道中的乙醇结合口袋进行结构功能分析 (1)，解析与乙醇复合的 GIRK 通道的高分辨率结构，以揭示乙醇依赖性门控中发生的通道蛋白的构象变化 (2)，并利用单通道记录和化学修饰，详细阐述 GIRK 通道的乙醇依赖性激活的机制模型 半胱氨酸取代的通道 (3)。这些实验的完成将揭示乙醇调节 GIRK 通道的结构基础，这将为了解乙醇调节其他类型离子通道的机制提供见解。了解乙醇调节离子通道的分子机制可能有助于开发治疗酒精依赖的新型药物，从而直接造福人类健康。